Penicillin salts of aminoacid esters of sterols and preparation thereof



UNITED STATES PATENT OFFICE PENICILLIN SALTS OF AMINOACID ESTERS OF STEROLS AND PREPARATION THEREOF Robert D. Coghill, Lake Bluif, Arthur W. Weston, Waukegan, and Donald W. MacCorquodale, Highland Park, 111., assignors to Abbott Laboratories, North Chicago, 11]., a corporation of lllinois No Drawing. Application February 20, 1947, Serial No. 729,888

This invention relates in general to organic salts of antibiotic compounds and specifically to insoluble salts of high molecular weight basic esters of amino carboxylic acids and acidic anti- 14 Claims. (01. zoo-439.5)

2 Rf are hydrogen or a lower alkyl group, and Z is an antibiotic residue such as that of a penicillin.

A general preparation of these compounds may biotic compounds. p 5 be accomplished in the following manner: A The object of this invention is to provide difhigh molecular weight hydroxy compound, such ficulty soluble compounds of antibiotics, such as as a sterol, or high aliphatic alcohol, is reacted the penicillins, which upon administration into with a haloacyl halide. The haloester formed is the body will maintain therapeutically effective then reacted with an amine to form the aminoconcentrations in the blood stream for a longer in ester. The aminoester, which is a base, may be period of time than the preparations of the prior reacted with an acid to form the salt for isolaart. The penicillins are found to be eliminated tion purposes. The free base (amine) dissolved from the body in varying degrees of rapidity, but in a solvent such as ether, and a solution of the in all cases the duration of action of the comacidic form of the antibiotic are mixed, and upon pound is for a relatively short period of time. standing and cooling, the salt of the amine base This period is usually limited to several hours. and the antibiotic crystallizes from the solution. This means that when penicillin is administered This synthesis may be described by the folby intravenous injection, the patient must lowing series of equations: usually be hospitalized so as to be available for O n 0 the penicillin therapy. In the case of'intramusg Y 1 cular injection, the Romansky formula using calcium penicillin has been successful in pro- 0 R o a longing blood levels in therapeutically efiective l,L l l; concentrations for a maximum of about six hours p in dogs. For the purpose of comparison, when 5 the compounds of this invention are incorporated 0 R o into a wax-oil medium-and injected into dogs. LY '+A A-0 -YN-Ae we have discovered that therapeutically effective I blood levels are maintained in the order of 16 o R 0 R to 20 hours or more. Therefore, when adminisg NaHCO; 1 tered in this manner, therapeutic levels may be maintained for the period of nearly a day with a single injection. In the case of human beings o 1'}. 0 this period may extend for several days.

The xplanation of this phenomenon appar- A 0 g Y N+Ho E z ently rests in the fact that since the penicillin 0 R o salts of this invention are insoluble in the fluids g of the body, the penicillin is released slowly by hydrolysis in the body, thus becoming available 1 for absorption into the blood stream in gradual amounts. Thus the dose of the penicillin is not 40 symbols used the.same meaning as dissipated due to rapid elimination from the gwen above In addmon x Palogen atom body slxilclzmasi chlorine, bromine, and iodine and Ac is The compounds of this invention are salts of a 1 high-molecular weight esters, such as the sterol 3: ifi given apove descrfbes the general esters of amino organic acids, and acidic antisyn es 5 of 1s P The examples that moths. These compounds are represented by follow will relateto specific examples of the invention for the purpose of illustration. the following structure.

J Cholcsterul chloroacetate 0 wherein A is a high-molecular weight radical -3 such as a sterol residue, Y is an aliphatic residue A mixture of 38.5 gms. of cholesterol and 11.3 containing from 1 to.- 11 carbon atoms, R and 55 sums. of chloroacetyl chloride in cc. of dry above.

toluene is refluxed tor rive hours, then cooled and the solid removed by filtration. A good yield of the product, cholesteryl chloroacetate, melting at 161-163 C., is obtained.

Using bromoacetyl chloride, chloroacetyl bromide or bromoacetyl bromide the productsobtained are cholesteryl bromoacetate, cholesteryl chloroacetate and cholesteryl bromoacetate, respectively. When iodacetyl chloride, bromide, and iodide are used, the product is cholesteryl iodoacetate.

Instead of using acetyl as the acyl group in the example above, other acyl groups may be used. For example, the halo-propionyl, -butyryl, -valeryl, -caproyl, -enanthylyl, -caprylyl, -pelargonyl, -capryl, -undecylyl or -laurylyl halides may be used. In which case, the compounds produced are cholesterylhalo-propionate, -butyrate, -valerate, -caproate, -enanthylate, -caprylate, -pelargonate, -caprate, -undecylate, and -laurate, respectively. The halogen may also be situated on any carbon of the alkyl chain and the chain itself may be straight or branched.

As examples of the branched chain haloacyl halides which may be used are the a-haloisobutyric acid and the fl-halobutyric acid. The compounds produced with these acid halides are cholesteryl-a-haloisobutyrate and cholesteryl-phaloisobutyrate.

In a straight chain halo acyl halide, such as halopropionyl halide, the halogen on the alkyl chain may be either in the alpha or beta position.

In the former case, the compound formed is cholesteryl a-halopropionate and in the latter case, the compound formed is cholesteryl-p-halopropionate.

The cholesterol used in the example may be replaced by other high molecular weight alcohols such as stigmasterol, sitosterol, phytosterol, ergosterol, calciferol, lumisterol, cetyl, ceryl. myricyl, and octadecyl alcohols. I

The esteriflcation in this example may be accomplished by other means than that described For example, the alcohol may be converted to the halide and the halide reacted with the metallic salt of the haloorganic acid. This may be illustrated by the following reactions where A, x, and Y are as represented above and M represents a metal such as sodium:

wherein W is chlorine or an amine forming group such as a nitro, cyano, formyl, or carboxyl group. The esteriflcation may also be carried out by reacting the alcohol with a haloorganic anhydride as illustrated below:

The hydroxy compound may be reacted with the haloorganic acid in the presence of an acid catalyst. The illustration of the reaction is given below:

EXAMPLE II cholesteryl diethylaminoacetate hydrochloride About 17.1 gms. of cholesteryl chloroacetate (Example I) is dissolved in 55 cc. of hot, dry benzene. About 5.4 gms. of diethylamine is added and the solution refluxed for twelve hours. The reaction'mixture is washed with water, then dried over anhydrous sodium sulfate. The addition of gaseous hydrogen chloride to the benzene solution precipitates the white, solid hydrochloride salt which is separated by filtration and washed with ether. The yield of product melting at 228-230 C. (with decomposition) is quantitative. By crystallizing from alcohol, pure material melting at 237 C. (with decomposition) is obtained.

In this example the diethylamine may be replaced with other secondary amines such as dimethyl, dipropyl and dibutyl. In the use of these amines the products produced are cholesteryldimethylaminoacetate, dipropylaminoacetate and dibutylaminoacetate hydrochlorides. The alkyl groups oi! the amine need not be the same and as an example to illustrate this, 11 methylethylamine is used, the product is cholesteryl methylethylaminoacetate hydrochloride.

Branched alkyl amines may be used as well as straight chain amines. For example, diisopropylamine and ethylisopropylamine may be used. In which case the compounds formed are cholesteryl diisopropylaminoacetate hydrochloride and cholesteryl ethylisopropylamine hydrochloride, respectively.

A primary amine or ammonia may be used instead or a secondary amine in which case the aminoester formed is monoallqlated. For example, ii ethyl amine, methyl amine or ammonia are used the compounds formed are cholesteryl ethylaminoacetate, methylaminoacetate and aminoacetate.

Instead or hydrogen chloride being used in the example above, other acids may be used. some or these acids are hydrobromic, sulfuric, nitric and acetic, which will give the dialkylaminehydrobromide, -suliate, -nitrate and -acetate, respectively.

In addition to the process described in Example II, the amino esters may be prepared by reacting the aminoacyl halide acid addition product with a suitable high molecular-weight alcohol. For example, the hydrochloride of diethylaminoacetyl chloride may be reacted with the cholesterol to give cholesteryl diethylaminoacetate hydrochloride as above.

EXAMPLE III cholesteryl diethillaminoacetate 0 01B: CnHcr-O- CHsN' About 5 gms. of cholesteryl diethylaminoacetate hydrochloride (Example 11) is stirred vigorously with an excess of a nearly saturated sodium bicarbonate solution and cc. oi ether until the solid disappears. The ether layer is then separated, the aqueous layer extracted with ether and the ether solutions combined. dried and concentrated. A residue of 4.6 gm. is obtained which on crystallization from alcohol gives 4.3 gm. or 92% of theoretical yield of prods uct melting at 88-90 O. The pure material melts at 90-91 C. I

Other neutralizing agents than sodium bicarbonate may be used to convert the amine acid methods of reduction, and when the group is carboxyl or formyl by reacting with an amine followed by reduction. I

These amino esters may be prepared by ester exchange. By way of example, cholesteryl diethylaminoacetate may be prepared by reacting cholesterol with the methyl ester of diethylaminoacetic acid. This reaction is illustrated by the following equation:

cholesteryl diethulaminoacetate salt of penicillin "G ether solution of the penicillin acid. The solution is stirred and then allowed to stand at room temperature.' Crystallization begins shortly. After placing the solution in the refrigerator over night, the crystals are collected by centrifugation or filtration, washed with anhydrous ether and dried, first in a stream of nitrogen and then under vacuum. The yield is 6.79 gins. (75.4%) of snow-white crystals. A second crop of about 1.11 ms. is obtained after concentration of'the mother liquors. The material is non-hygroscopic andrmelts to a colorless liquid at l04-107 C. The compound is almost completely insoluble in water, very slightly soluble in peanut oil and in ether, and very soluble in chloroform,

When analysed by the conventional methods of microchemical analysis the compound is found to contain 70.31% carbon, 8.76% hydrogen and 4.96% nitrogen. These results agree with the calculated percentages for CmH'rsOsNsS which are 70.70% carbon, 9.00% hydrogen, 5.05% nitrogen. Bioassay of the compound (by releasing the penicillin with a buifer solution) indicates 715 Oxford units of penicillin G per milligram. The theoretical potency contributed by the penicillin component in this molecule is 710 units per miiliram.

1 In the above example other acid antibiotics common penicillins may be used. These peni-.

cillins have been given the names of A -pentenyl-, p-hydroxybenzyl-, and n-heptylpenicillins respectively in the Ofllcial Penicillin Monograph.

0 c H 0 H IL l 5 The compounds produced by using these penicillins in forming salts with the cholesteryl di- H 1H5 ethylaminoacetate are: A -pentenyl-, p-hydroxy- H benzyland n-heptylpenicillinates of cholesteryl Q diethylaminoacetate. H30 5 0 About 4 gms. of pure crystalline sodium salt EXAMPLE v ofpenicillin G (which has been assigned the cholesteryl diethylaminoacetate salt of penichemical name sodium benzylpenicillinate in the cillin F I O CaHl CiHs H flHir-0 -CHaN-OE .NC=O

CHr -l-n-.c-ca,-cn=cn-cm-cm In accordance with the procedures set forth above, the cholesteryl diethylaminoacetate salt of penicillin F, (n -pentenylpenicillin) may be prepared by reacting cholesteryldiethylaminoe acetate with penicillin 1 1" EXAMPLE VI 00 cholesteryl diethylaminoacetate salt of penicillin X" tilled water and concentrated to 20 cc. volume in In accordance with the procedures set forth a current of nitrogen.

About 5 gins. of the pure cholesteryl diethylaminoacetate base (Example III) is dissolved'in 20 cc. of anhydrous ether. To this 20 cc. ether above, the cholesteryl diethylaminoacetate salt of penicillin X (p-hydroxybenzylpenicillin) may be prepared by reacting cholesteryldiethylsolution of the base, is added slowly the 20 cc. aminoacetate with penicillin "X."

emails mMPLEVH cholesteryl diethylaminoacetate salt I cillin "rt" can; cam CflHf-O-LCHI OCb-N- =0 of peni- By using the procedure set forth in Example IV using penicillin "K (n-heptylpenicillin) with the cholesteryl diethylaminoacetate the above compound is produced.

Other oi. the less common penicillins may be used such as those produced by precursor activity or iermentations of other penicillin producing molds. As examples of those produced by precursor activity which may be used are phenylmercaptomethyl-, p bromophenylmercaptomethyl-, benzylmercaptomethyl-, phenylethylmercaptomethyl-, and p-hydroxyphenylmercaptomethylpenicillins. Tnese compounds are disclosed in U. S. Patent Application No. 721,146. when these penicillins are used in salt formation with the cholesteryl diethylaminoacetate the products obtained are: phenylmercaptomethylpenicillinate, p bromophenylmercaptomethylpenicillinate, benzylmercaptomethylpenicillinate, phenylethylmercaptomethylpeniciilinate, and phydroxypherwlmercaptomethylpenicillinate of cholesteryl diethylaminoacetate. As examples of those penicillins produced naturally by the fermentation of other penicinin molds are gigantic acid which is n-pentylpenicillin (dihydropenicillin F) and flavicidin which is isomeric with penicillin F (the unsaturation in the pentyl group is in a difierent position). When used for 'salt formation with cholesteryl diethylaminoacetate the products obtained are the cholesteryl diethylaminoacetate salt of gigantic acid and the cholesteryl diethylaminoacetate salt of flavicidin.

Other acid antibiotics which may be used instead of the penicillins are: penicillic acid, citrinic acid, and aspergillic acid.

In combination with the penicillins described above other bases may be used such as those described in Example III or bases prepared by the processes of the invention starting with the compounds described in Examples I and II. Included in this group are benzylpenicillinate of cholesteryl dimethylamino-n-propionate; A pentenylpenicillinate o1 cholesteryl dipropylaminolaurate; p-hydroxybenzylpenici1linate of cholesteryl dibutylammo-n-butyrate; p-hydroxyphenylmercaptomethylpenicillinate of cholesteryl diethylamino-n-butyrate; benzylmercaptomethylpenicillinate of cholesteryl dimethyiaminoacetate; benzylpenicillinate of stigmasteryl diethylaminoacetate; A -pentenylpenicillinate of ergosteryl diethylaminoacetate; benzylpenicillinate o1 ergosteryl diethylaminoacetate; benzylpenicillinate of cetyl methylethylaminovalerate; n-heptylpenicillinate oi. cetyl dimethyiaminoiso-butyrate; aspergillate or n-octadecyl di-npropylaminoacetate; p-hydroxyphenylmercaptomethylpenicillinate of n-octadecyl methyl-npropylaminopropionate; phenyl mercaptomethylpenicillinate of stigmasteryl diethylaminoacetate; n-heptylpenicillinate of stigmasteryl dimethylamino-n-butyrate; p-hydroxybenzylpenicillinate of stigmasteryl dimethylaminocaprate; penicillic acid salt of calciteryl diethylaminoa laurate; benzylpeniciilinate of ca1ciferyldicthyl= aminoacetate; A -pentenvlpenicillinate of calcii'eryl diethylaminoacetate; p-hvdroxybenzylpenicillinate of calciieryl diethylaminoacetate; n-heptylpenicillinate of calciteryl diethylaminoacetate; benzylpenicillinate oi lumisteryl diethylaminoacetate; A -pentenylpenicillinate of lumic= teryl diethylaminoacetate; A -pentenylpenicillinate of stigmasteryl diethylaminoacetate; phydroxybenzylpenicillinate oi stigmasteryl diethylaminoacetate; p-hydroxybenzylpeniclllinate of lumisteryl diethylaminoacetate; p-hydroxybenzylpenicillinate of ergosteryl diethylaminoacetate; benzylpenicillinate of cetyl diethylaminoacetate; A -pentenylpenicillinate of cetyl diethylaminoacetate; p-hydroxypeniciliinate oi cetyl diethylaminoacetate; n-heptylpenicillinate o1 cetyl diethylaminoacetate; n-heptylpenicillinate of ergosteryl diethylaminoacetate; benzyipenicillinate oi octadecyl diethylaminoacetate; .fi-pentenylpenicillinate of octadecyl diethylaminoacetate; n-heptylpenicillinate of octadecyl diethylar'ninoacetate; p-hydroxybenzylpenicillinate of octadecyl dlethylaminoacetate; benzylpenicillinate of ,sitosteryl diethylaminoacetate; A -pentenyipenicillinate of sitosteryl diethylaminoacetate; p-hydroxypenicillinate of sitosteryl diethvlaminoacetate; n-heptylpenicillinate,

pentenylpenicillinate of cholesteryl aminoacetate; benzylpenicillinate of phytosteryl diethylaminoacetate; n-heptylpenicillinate 01' phytosteryl diethylaminoacetate; p-hydroxybenzylpenicillinate of phytosteryl diethylaminoacetate; A -pentenylpenicillinate of phytosteryl diethylaminoacetate and benzylpenicillinate of phytosteryl ethylaminoacetate.

In addition to the process outlined above these amine base salts of antibiotics may be prepared by reacting the amine acid addition product (Example 11) with a salt such as the sodium salt of the antibiotic acid. This process may be illus trated by the following reaction:

wherein A is a high molecularweight radical, such as a sterol residue, Y is an aliphatic residue containing from 1 to ll carbon atoms, R and R are hydrogen or a lower alkyl group, B is a cation, An is an anion. and Z is an antibiotic residue such as that of a penicillin.

Salts of antibiotic compounds according to the invention may be made up into a variety of pharmaceutical vehicles for the administration of these therapeutic compounds. Among the compositions of matter which have been prepared and used successfully are aqueous suspensions, saline suspensions, vegetable oil suspensions, vegetable oil-wax suspensions, and tablets, etc.

per cc. of wax-oil mixture. The concentration physical properties to the mass.

EXAMPLEXI As a means of out administration of the antibiotic salts of this vention is the incorporation of therapeutically significant amounts of the medicament into a conventional tablet base and forming tablets by the usual means. These tablets may be provided with enteric coating if desired.

The alkyl groups on the amine preferably con- Because of the slow liberation of the antibiotic. tam from one to four carbon atoms compound into the blood stream,v it is possible The term halogen unless specifically defined to maintain a longer duration of a therapeutieludes chlorine bromine and iodhm cally significant concentration of the antibiotic The h mo1ecu1ar wight alcohols and radi by the administratmn these Pharmaceutical 5 cals refe ed to are intended to contain at least preparatiomsixteen carbon atoms.

As illustrative examples of the preparations ot may readily adapt the invention for referred to above are the following! use under varying conditions of service, by em- EXAMPLE vm ploying one or more of the novel features disclose r i r I These insoluble antibiotic salts form aqueous y 1: 2 :3 3; g g g ggz gg :3 suspensions and may be prepared by adding the invention, we desire to claim the following subdesired quantity of the compound to a suitable matten voluaie of sterile distilled water or isotonic saline we claim. 1. The penicillin salt of a steryl ester of an EXAMPLE Ix amino lower fatty acid selected from the group Suspensions of the insoluble antibiotic salts of consisting of N'Hz-alkylene-COOH; lower althis invention in vegetable oils, such as peanut oil, kyl-NH-alkylene-COOH; and (lower alkyl);-N- have been made. For example. by making a satualkylene COOH, said alkylene group having 1-11 rated solution of the cholesteryl -diethy1aminocarbon atoms. acetate salt of benzylpeniciiiinate (penicillin 2. The penicillin salt of a steryl ester of an "6") (Example IV) in sterile peanut oil at room amino acetic acid repr wented by the formula temperature, a bioassay of the product indicates NHzCHzCOOH. a potency of 874 Oxford units per cc. Since the 3. The penicillin salt of a steryl ester of a potency of this penicillin salt is715 units per J5 lower-alkylamino acetic acid. milligram, this indicates a concentration of about 4. The penicillin salt of a steryl ester of a di- 1.22 mg. of material per cc. of oil. The oil has lower-alkylamino acetic acid. the property of retarding absorption of the 1 5. The penicillin salt of the cholesteryl ester of compounds of the present invention into the an amino acetic acid represented by the formula blood stream. a NHzCHzCOOI-I.

EXAMPLE X t 6. The penicillin salt of the cholesteryl ester We have discovered that a very successful form of lower'alkylammo acetic acid for administering these antibiotic salts is by The penmmiln salt of the cholesteryl ester means of a suspension in a wax-vegetable oil ofa'di'lower'alky amino acetic acld' medium This type of preparation has a very 8. The penicillin salt of the cholesteryl ester prolonged period of action since it combines two of diethylamino i 1 1 1 insolubilities. Therefore, the antibiotic is made The compound 0 0 es ery d ethy aminoace' available for absorption into the blood stream in tate salt of benzyl penicmmate which has the even more graduated amounts. An advantageous following structure: method for preparation of this type of pharma- E Gin ciHsH ceutical product is to mix a desired amount of the insoluble antibiotic salt with the sterile oil and o CHPN O F N C 0 then molten wax such as beeswax is added. The l l mixture is allowed to cool while continuing the 011,- NC-CH: stirring so as to solidify the mass. Various conch: 8 iii 1!! g centrations of the components of the mixture 1 may be used but usually the concentrations of 10. The compound cholesteryl diethylaminothe insoluble antibiotic salt are fairly high; in acetate salt of A -pentenylpenicillin which has the order of 200,000 or 300,000 units of penicillin the following structure:

' O CzHs C|H| H CnHuO-PJ-CH: NO'GJJ NC=O CHn-v L-JZ-N-C-CHs-CH=CHCH2CHI or. no i i t 1 11. The compound cholesteryl diethylaminoacetate salt of p-hydroiwbenzylpenicillm which has the following structure:

12. The compound cholesteryl diethylaminoacetate salt of n-heptylpenicillin which has the following structure:

OHrlNC-C H;r-n

Ce. s A 1i 1 ii 18. The process of preparing a salt of claim 1 which comprises mixing penicillin as the free acid with asteryi ester of the amino lower fatty acid in an inert organic solvent.

14. The process 01 preparing the salt of claim 9 which comprises mixing penicillin G as the free acid with cholesteryl diethyiaminoacetate in ether.

ROBERT D. COGHll-L. ARTHUR W. WESTON.

DONALD W. MAcCORQUODALE.

12 nm'nnsncns crrm The following references are of record in the file of this patent:

OTHER REFERENCES nobty et 9.1.. Proc. Soc. Exp. Biol. & Mei. June 1942, page 287.

15 Beyer at 0.1., Science. vol. 100, Aug. 4, 1944,

.page 107.

J. Amer. Med. Assoc, July 21, 1945, page 910. Greet et al., The Pharmaceutical Journal. Sept. 23, 1944, page 124.

go Krantz et al.. Science." vol. 101, Jan. 15, 1945,

page 518. 

1. THE PENICILLIN SALT OF A STERYL ESTER OF AN AMINO LOWER FATTY ACID SELECTED FROM THE GROUP CONSISTING OF NH$-ALKYLENE-COOH; LOWER ALKYL-NY-ALKYLENE-COOH; AND (LOWER ALKYL)2-NALKYLENE COOH, SAID ALKYLENE GROUP HAVING 1-11 CARBON ATOMS.
 9. THE COMPOUND CHOLESTERYL DIETHYLAMINOACETATE SALT OF BENZYL PENICILLINATE WHICH HAS THE FOLLOWING STRUCTURE: 